Method for identifying the chemical structure of lipids and ion mobility spectrometry tandem mass spectrometer

Abstract

To relate to a mass spectrometry field and particularly to provide a method for determining a chemical structure of a lipid and an ion mobility-tandem mass spectrometer.SOLUTION: A method for determining a chemical structure of a lipid includes: an ionization step of ionizing a sample to obtain sample ions; an ion mobility-based separation step of separating target lipid ions from the sample ions, based on ion mobility; a first dissociation step of dissociating a target lipid ions with dissociation energy adjusted to break a first chemical bond of the target lipid ions; a mass-based selection step of selecting the target lipid ions, whose first chemical bond is broken, based on a mass number to obtain fragment ions; a second dissociation step of dissociating the fragment ions to at least break a second chemical bond of the fragment ions which has bond energy higher than the first chemical bond, to obtain diagnostic ions; and a mass analysis step of performing a mass analysis on the diagnostic ions.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to the field of mass spectrometry, and more particularly to a method for identifying the chemical structure of lipids and an ion mobility spectrometry tandem mass spectrometer. [Background technology]

[0002] Lipids are important nutrients and components of living cells, and are closely related to some important immune functions and metabolic disorders. Currently, the Lipid Metabolites and Pathways Strategy (LIPID MAPS) has been launched to promote lipid omics research by establishing a classification database.

[0003] Complete lipid labeling and identification information includes classification, elemental composition, size and position of R-groups (sn-positions), number and position of double bonds, and cis-trans isomeric orientation of double bonds. For unsaturated lipids with a glycerol backbone, such as glycerides and glycerophospholipids, the sn-positions in the fatty chain containing carbon-carbon double bonds must also be identified.

[0004] Multi-stage tandem mass spectrometry plays an important role in the structural analysis of compounds. Hsu and Turk proposed a pseudo-tertiary (MS3) tandem mass spectrometry method that can identify the sn-position of glycerophospholipids. First, they used in-source CID to obtain a high-abundance headgroup-free fragment ion signal [M+Li-183]. +The fragment ions are then subjected to collision-induced dissociation to generate sn-diagnostic ions (Non-Patent Document 1). Non-Patent Document 2 proposed a combined MS3 method combining hybrid collisional activation and UV-visible spectrophotometry to simultaneously identify double bond and sn-positions in glycerol-based unsaturated lipids with a single sample injection, but the abundance of diagnostic ions remains to be improved. These pseudo-MS3 and MS3 analytical methods are not suitable for isomer selection and analysis. Furthermore, when multiple ions are selected in the first stage using quadrupole mass spectrometry, the non-target ions are lost, resulting in a low overall duty cycle. In addition to directly performing multistage tandem mass spectrometry on compounds, chemical derivatization can be used to pre-modify analytes to improve ionization efficiency, enhance structural differences, and improve chromatographic behavior, and then perform multistage tandem mass spectrometry on the derivatives. Ma et al., in Non-Patent Document 3, used charge-label derivatization and MS3 to accurately identify the C=C and sn-positions in derivatized glycerophospholipids. The method provided by the paper requires mass selection of specific parent ions using an ion trap mass spectrometer, and the duty ratio and resolution are limited by the ion trap mass spectrometer.

[0005] Ion mobility spectroscopy (IMS) can achieve high-throughput separation and analysis of isomers under gas-phase conditions. When combined with mass spectrometry, it offers high resolution and has effects similar to multistage tandem mass spectrometry (NPL 4; NPL 5). This allows detailed structural information of analytes to be obtained, making it widely used for structural identification in metabonomics, glycomics, and proteomics. For example, numerous studies have demonstrated that the combination of cyclic ion mobility (cIM) and collision-induced dissociation (CID), such as cIM-CID-cIM and cIM-CID-cIM-CID-cIM modes, can achieve separation and in situ measurement of carbohydrate isomers (NPL 6; NPL 7). Bleiholder's team proposed a combination of trapped ion mobility (TIMS)-based tandem mobility spectrometry and tandem mass spectrometry, which incorporates mobility selection and collisional activation (Patent Literature 1, Non-Patent Literature 8), enabling structural identification of polypeptides and proteins (NPL 9; NPL 10), and sugars and their isomers (NPL 11). Nicholas B. Borotto et al. have realized pre-transfer collision-induced unfolding based on TIMS, which allows rapid identification of protein conformations (Non-Patent Document 12). Furthermore, pre-transfer collision-induced unfolding can be further combined with tandem mass spectrometry to achieve accurate protein sequencing (Non-Patent Document 13).

[0006] In recent years, ion mobility mass spectrometry has also achieved good results in the in-depth identification of lipid structures (Non-Patent Document 14; Non-Patent Document 15; Non-Patent Document 16). Baker and colleagues proposed a lipid omics analysis method combining reversed-phase liquid chromatography and ion mobility mass spectrometry, which can separate lipids and their isomers in three dimensions: analyte polarity, structure, and mass-to-charge ratio magnitude, thereby increasing peak capacity. Liquid chromatography can separate different types of lipids, and different types of lipids form different ion trend lines in ion mobility mass spectrometry. Furthermore, crude separation of different lipid subclasses can be observed in ion mobility spectra (Non-Patent Document 17). Due to instrument resolution limitations, different lipid isomers only achieve shoulder peak separation. F. Fernandez-Lima et al. used high-resolution TIMS under specific instrument parameters (average resolution exceeding 320) to identify double bond positional isomerism in phosphatidylcholine sodium ion adducts and cis-trans double bond isomerism in proton adducts, respectively. Furthermore, if the instrument parameters meet the requirements for ultrahigh resolution (over 410), this method can be used to identify sn-positional isomers (Non-Patent Document 18). However, the resolving power of most commercial ion mobility instruments is less than 200, making it difficult to meet these requirements. Forming metal ion adducts contributes to improved isomer resolution. M. Groessl et al. used drift-time ion mobility mass spectrometry to distinguish between positional isomerism of the double bond in a silver ion adduct of phosphatidylcholine, as well as cis-trans isomerism and sn-isomerism of the double bond (Non-Patent Document 19). Similarly, Yan et al. used drift-time ion mobility mass spectrometry to separate and analyze cis-trans isomers of the carbon-carbon double bond in a monovalent copper ion adduct of phosphatidylcholine, but the resolution was poor (Non-Patent Document 20).

[0007] Many methods for identifying lipid isomers based on ion mobility spectroscopy have been developed, but these methods require the use of standard samples as references, making it difficult to analyze the structures of unknown compounds. Furthermore, the resolution of ion mobility spectroscopy is inferior to that of mass spectroscopy, limiting the analysis of complex substrate samples. Combining the separation capabilities of ion mobility with the resolving power of tandem mass spectrometry offers significant potential for the structural analysis of unknown lipid compounds in biological samples. Brodbelt's team used UVPD and drift-time ion mobility mass spectrometry to measure lipid isomer collision cross sections and identify lipid double bond and cyclopropane positions (Non-Patent Document 21). However, while this method is suitable for polar lipid analysis, it struggles to detect lipids with medium and low polarity. Xia et al. combined the PB reaction with a trapped ion mobility tandem mass spectrometry system to achieve the separation and analysis of conjugated fatty acid isomers. Combining ion mobility spectrometry with tandem mass spectrometry allows accurate assignment of mobility peaks and double bonds, even in the absence of standard samples. However, the reaction products of PB reactions with conjugated fatty acids are diverse, and the resulting mobility spectra are highly complex, limiting their application in complex mixtures (Non-Patent Document 22). In 2023, Xia's team combined liquid chromatography, ion mobility spectroscopy, and PB reaction tandem mass spectrometry to establish a series of in-depth lipid structure analysis procedures, enabling stepwise identification of double bond positions and sn-positions. This was successfully applied to lipid omics analysis in biological samples such as bovine liver and cells, enabling more complete lipid profile information to be obtained (Non-Patent Document 23). However, the step-by-step pretreatment and multiple-batch analysis process not only reduces analytical throughput but also results in sample loss. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] US10794861 B2 [License 2] Chinese Patent CN113495112A [Non-licensed literature]

[0009] [Non-licensed Document 1] J.Am.Soc.Mass Spectrom.2003, 14, 352 [Non-licensed Document 2] Brodbelt et al, "Pinpointing Double Bond and sn-Positions in Glycerophospholipids via Hybrid193nm Ultraviolet Photodessociation (UVPD) Mass Spectrometry" (J.Am.Chem.Soc.2017, 139, 15681-15690) [Non-licensed Document 3] Ma et al, "Large-scale lipid analysis with C=C location and sn-position isomer resolving power" (Nat. Commun., 2020, 11, 375) [Non-licensed Document 4] Biochem.Soc.T.2020, 48, 2457 [Non-licensed Document 5] Anal. Chem. 2006, 78, 4161 [Non-licensed Document 6] Anal.Chem.2021, 93, 6254 [Non-licensed Document 7] Annual Rev.Anal.Chem.2023, 16, 27 [Non-licensed Document 8] Analyst2022, 147, 2317 [Non-licensed Document 9] Analyst2018, 143, 2249 [Non-licensed Document 10] J.Am.Soc.Mass Spectrom.2023, 34, 2247 [Non-licensed Document 11] Anal.Chem.2023, 95, 747 [Non-Patent Document 12] J.Am.Soc.Mass Spectrom.2022, 33, 83 [Non-Patent Document 13] J.Am.Soc.Mass Spectrom.2023, 34, 2232 [Non-Patent Document 14] J.Chromatogr.A, 2017, 1530, 90 [Non-Patent Document 15] J. Sep. Sci. 2018, 41, 20 [Non-Patent Document 16] Front.Mol.Biosci.2023, 16, 10, 1112521 [Non-Patent Document 17] Analyst, 2016, 141, 1649 [Non-Patent Document 18] Anal.Chem.2019, 91, 5021 [Non-Patent Document 19] Analyst, 2015, 140, 6904 [Non-Patent Document 20] Int.J.Mass Spectrom.2022, 479, 116889 [Non-Patent Document 21] Anal.Chem.2022, 94, 4252 [Non-Patent Document 22] Anal.Chem.2019, 91, 7173 [Non-Patent Document 23] Nat.Commun.2023, 14, 4263 Summary of the Invention [Problem to be solved by the invention]

[0010] In response to the above problems, the present invention provides a method for identifying the chemical structure of lipids and an ion mobility spectrometry tandem mass spectrometer that increases the abundance of diagnostic ions and has good duty ratio and resolution. [Means for solving the problem]

[0011] A first aspect of the present application is a method for detecting a sample using an ionization step of ionizing a sample to obtain sample ions; an ion mobility separation step in which target lipid class ions are separated from sample ions based on ion mobility; a first dissociation step of dissociating the target lipid ions with a dissociation energy suitable for cleaving a first chemical bond of the target lipid ions; A mass selection step of selecting target lipid ions whose first chemical bond has been cleaved based on their mass numbers to obtain fragment ions; a second dissociation step of dissociating the fragment ions and cleaving at least a second chemical bond of the fragment ions, the second chemical bond having a higher bond energy than the first chemical bond, to obtain diagnostic ions; and performing mass spectrometry on the diagnostic ions.

[0012] The lipids are unsaturated lipids having carbon-carbon double bonds in the fatty chain, and the method may be used to identify the position of the carbon-carbon double bond in the fatty chain and the position of sn in the fatty chain.

[0013] The method may further include a derivatization reaction step of labeling the carbon-carbon double bond by a derivatization reaction prior to the ionization step.

[0014] The lipids may be phospholipids or sphingolipids, the first chemical bond being a bond to a polar head group of a phospholipid or a polar head group of a sphingolipid, and the second chemical bond being a chemical bond obtained by a derivatization reaction of a carbon-carbon double bond.

[0015] The derivatization reaction may be an aziridination reaction, an epoxidation reaction, a Paterno-Buchi reaction, a singlet oxygen-ene reaction or a Diels-Alder reaction.

[0016] The lipids may be fatty acyl, glycerides, glycerophospholipids, sphingolipids, sterol esters, pregnenolone lipids, glycolipids or polyketides.

[0017] The method may further include a first pre-scan step in which mass analysis is performed on sample ions that have not undergone the first dissociation step and the second dissociation step.

[0018] The method may further include a second pre-scan step in which mass analysis is performed on the sample ions that have only been dissociated once.

[0019] A second aspect of the present application further provides an ion mobility spectrometry tandem mass spectrometer, including an ion source, an ion mobility spectrometer, a first dissociation device, a mass filter, a second dissociation device, and a mass analyzer. The ion source ionizes a sample to obtain sample ions. The ion mobility spectrometer separates target lipid ions from the sample ions. The first dissociation device dissociates the target lipid ions, with the dissociation energy of the first dissociation device being suitable for cleaving a first chemical bond of the target lipid ions. The mass filter selects the target lipid ions whose first chemical bond has been cleaved to obtain fragment ions. The second dissociation device dissociates the fragment ions and cleaves at least a second chemical bond of the fragment ions, the bond energy of which is higher than that of the first chemical bond, to obtain diagnostic ions. The mass analyzer performs mass analysis on the diagnostic ions.

[0020] The first dissociation device may be a collision-induced dissociation device, and the terminal electrode voltage of the first dissociation device may be 10-70 eV. The second dissociation device may be a collision-induced dissociation device, and the dissociation energy of the second dissociation device may be 30-70 eV. [Effects of the Invention]

[0021] <Useful effects> This method utilizes ion mobility to select various target lipid ions at a high duty ratio and then performs subsequent multiplexed target tandem mass spectrometry to reduce the complexity of a single spectrogram. This method involves performing a pseudo-multistep collision-induced dissociation (PSD) technique, in which a first chemical bond with a relatively weak binding energy in the target lipid ions is cleaved to remove interfering groups linked to the target lipid ions via the first chemical bond. The fragment ions from which the interfering groups have been removed are then selected and subjected to secondary dissociation. This method improves the mass spectrometry signal intensity of the diagnostic ions ultimately generated and alleviates or avoids the problem of increased spectrogram complexity due to the appearance of spectral peaks related to interfering groups in the final mass spectrogram. This results in a simpler spectrogram, making it easier to analyze and determine the chemical structures of lipids. [Brief explanation of the drawings]

[0022] [Figure 1] 1 is a flow chart of a method for identifying the chemical structures of lipids according to an embodiment of the present invention. [Figure 2] FIG. 1 is a schematic diagram of a system for implementing a method for identifying the chemical structures of lipids according to an embodiment of the present invention. [Figure 3] 1 is a schematic diagram of the structure of a preferred ion mobility spectrometry tandem mass spectrometer according to an embodiment of the present invention. [Figure 4] FIG. 2 is a diagram showing a specific reaction process of PC (18:1 / 16:0) according to the present embodiment. [Figure 5] FIG. 1 is a diagram illustrating the principle behind why the sn position of a carbon-carbon double bond can be identified from diagnostic ions with mass numbers m / z=290 or 360 [M+Na+]. [Figure 6] FIG. 1 is a comparison diagram of measurement results obtained by a general tandem mass spectrometry (MS2) method in the prior art and measurement results obtained by a method according to an embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0023] The technical solutions in the embodiments of the present invention will be described below clearly and completely in accordance with the drawings in the embodiments of the present invention, but it is clear that the described embodiments are only a part of the embodiments of the present invention, and are not all of the embodiments. Based on the embodiments of the present invention, all other embodiments that can be obtained by those skilled in the art without creative efforts fall within the protection scope of the present invention.

[0024] The method for identifying the chemical structure of lipids according to the present invention can be applied to identify lipids with interfering groups (e.g., polar head groups), which may be, for example, fatty acyl, glycerides, glycerophospholipids, sphingolipids, sterol esters, pregnenolone lipids, glycolipids, or polyketides.

[0025] In the method for identifying the chemical structure of lipids according to this embodiment, first, a first chemical bond with a relatively weak binding energy in the target lipid ion is cleaved to remove interfering groups linked via the first chemical bond in the target lipid ion, and then fragment ions from which the interfering groups have been removed are selected and subjected to secondary dissociation. This method improves the mass spectrometry signal intensity of the diagnostic ions finally generated, and alleviates or avoids the problem of increased spectrogram complexity due to the appearance of spectral peaks related to interfering groups in the final mass spectrogram. As a result, the spectrogram is simplified, making it easier to analyze and determine the chemical structure of lipids.

[0026] Fig. 1 is a flowchart of a method for identifying the chemical structures of lipids according to a first embodiment of the present invention. Referring to Fig. 1, the method according to this embodiment includes an ionization step S1, an ion mobility separation step S2, a first dissociation step S3, a mass selection step S4, a second dissociation step S5, and a mass analysis step S6, which are performed sequentially.

[0027] In the ionization step S1, the sample is ionized to obtain sample ions.

[0028] In the ion mobility separation step S2, target lipid class ions are separated from sample ions based on ion mobility.

[0029] In a first dissociation step S3, the target lipid ions are dissociated with a dissociation energy suitable for breaking the first chemical bond of the target lipid ions.

[0030] The first dissociation of the target lipid ion, i.e., the first dissociation step S3, can selectively cleave chemical bonds in the target lipid ion that have weak binding energy and may ultimately generate interference signals (i.e., the first chemical bonds that bind to interference groups).

[0031] The first dissociation step S3 is a selective dissociation step, i.e., a step in which the integrity of other chemical bonds (main chain chemical bonds, particularly the second chemical bond) is maintained as much as possible when the first chemical bond is cleaved. Specifically, to meet the above requirements, the dissociation energy can be set slightly higher than the threshold at which the first chemical bond can be cleaved. In some embodiments, when the first chemical bond has the lowest binding energy in the target lipid ion, the dissociation energy can be set to primarily cleave the first chemical bond while maintaining the integrity of the other chemical bonds to the maximum extent possible.

[0032] By setting the dissociation energy to be equal to or greater than the threshold at which the first chemical bond can be broken, the first chemical bond can be broken selectively, thereby eliminating interference of the interfering group with the final mass spectrometry test results while minimizing loss of ion abundance.

[0033] In the mass selection step S4, target lipid ions whose first chemical bond has been broken are selected based on the mass number to obtain fragment ions.

[0034] By selectively cleaving the first chemical bond, at least one pair of ions can be generated, one of which has an interfering group (e.g., a polar head group) and the other of which does not have the interfering group. The fragment ions selected in the mass selection step S4 are fragment ions that are linked by the first chemical bond and have the interfering group eliminated, i.e., ions that do not have the interfering group.

[0035] In a second dissociation step S5, the fragment ions are dissociated and at least second chemical bonds of the fragment ions, the second chemical bonds having higher bond energy than the first chemical bond, are broken to obtain diagnostic ions.

[0036] The resulting fragment ions free of interfering groups are further dissociated by secondary dissociation, i.e., the second dissociation step S5. The second dissociation step S5 can be selective or non-selective. However, in either case, because the interfering groups have already been removed in the first dissociation step S3 and the mass selection step S4, the mass spectrogram will not contain excessive spectral peaks associated with interfering groups. Therefore, the mass spectrogram will have fewer spectral peaks and the mass peaks of the diagnostic ions will be more intense.

[0037] Typically, when the same type of dissociation device is used, the dissociation energy employed in the second dissociation step S5 is higher than that employed in the first dissociation step S3, thereby enabling chemical bonds with higher bond energies to be broken.

[0038] In the mass analysis step S6, the diagnostic ions are subjected to mass analysis.

[0039] <Ion mobility spectrometry tandem mass spectrometer> Fig. 2 is a schematic diagram of a system for carrying out the method for identifying the chemical structures of lipids according to this embodiment, and Fig. 3 is a schematic diagram of the structure of a preferred ion mobility spectrometry tandem mass spectrometer according to this embodiment.

[0040] The ion mobility spectrometry tandem mass analyzer includes, in series, an ion source 1, an ion mobility spectrometer 2, a first dissociator 3, a mass filter 4, a second dissociator 5, and a mass analyzer 6. One or more ion optical devices 7 may further be connected between the above components to focus, conduct, or transport ions.

[0041] <Ion source> The ion source 1 performs an ionization step S1 to ionize the sample to obtain sample ions.

[0042] Ion sources include electrospray ionization sources (ESI), atmospheric pressure photoionization sources (APPI), atmospheric pressure chemical ionization sources (APCI), matrix-assisted laser desorption ionization sources (MALDI), laser desorption ionization sources (LDI), atmospheric pressure ionization sources (API), desorption ionization sources on silicon (DIOS), electron impact ionization sources (EI), chemical ionization sources (CI), field ionization sources (FI), field desorption ionization sources (FD), inductively coupled plasma ion sources (ICP), fast atom bombardment ion sources (FAB), liquid secondary ion mass spectrometry ion sources (LSIMS), and electrospray desorption ionization sources (D The ion source may be one selected from the group consisting of an ESI (electrospray ionization source), a nickel-63 radioactive ion source, an atmospheric pressure matrix-assisted laser desorption ionization source, a thermal spray ionization source, an atmospheric sampling glow discharge ionization source (ASGDI), a glow discharge ionization source (GD), an impactor ionization source, a real-time direct analysis ionization source (DART), a laser spray ionization source (LSI), an acoustic wave spray ionization source (SSI), a matrix-assisted entrance ionization source (MAII), a solvent-assisted entrance ionization source (SAII), a Penning ionization source, a laser ablation electrospray ionization source (LAESI), and a He plasma ionization source (HePI). Preferably, the ion source 1 is an electrospray ionization source, a nanospray ionization source, a desorption electrospray ionization source, an atmospheric pressure chemical ionization source, an atmospheric pressure photoionization source, or a matrix-assisted laser desorption ionization source. In this embodiment, the ion source is preferably an electrospray ionization source. In this embodiment, the ion source is preferably an electrospray ionization source.

[0043] <Ion mobility spectrometer> The ion mobility spectrometer 2 performs a mobility separation step S2 to separate the sample ions based on differences in ion mobility, and separates the target lipid ions from the sample ions.

[0044] The ion mobility spectrometer 2 includes one ion mobility spectrometer selected from the group consisting of a drift tube ion mobility spectrometry (DTIMS), a differential mobility analysis (DMA) device, a field asymmetric-waveform ion-mobility spectrometry (FAIMS) device, a traveling wave ion mobility spectrometry (TW-IMS), a differential mobility spectrometry (DMS) device, a transverse modulation ion mobility spectrometer, a trapped ion mobility spectrometer (TIMS), and a U-shaped ion mobility analyzer (UMA).

[0045] In this embodiment, the ion mobility spectrometer 2 is preferably a U-type ion mobility spectrometer, more preferably a U-type ion mobility spectrometer operating in a filtering mode. Patent Document 2 can be referenced for a description of the device structure of a U-type ion mobility spectrometer and the filtering mode (also referred to as "filter mode") applicable to this identification method.

[0046] The filtering mode filters out non-target ions and retains target ions. It also allows target ions to pass through the filter while maintaining their movement along a predetermined path. In other words, the filtering mode allows a continuous target ion stream to be output without changing the ion stream shape. Since the filtering mode does not allow ions to be locally present or stored, it is possible to avoid the loss of low-abundance ions due to space charge effects, making it highly suitable for lipid omics analysis research.

[0047] The ion mobility spectrometer 2 can provide a second dimension of data for tandem mass spectrometry. Isomers can be differentiated based on differences in ion mobility. In some embodiments, ion mobility spectra can be used to further distinguish the position of carbon-carbon double bonds or cis-trans isomer orientation and sn-isomer differentiation.

[0048] <First dissociation device> The first dissociation device 3 performs a first dissociation step S3, in which the dissociation energy of the first dissociation device 3 is set so as to cleave the first chemical bond with low binding energy (e.g., polar head group) in the target lipid ion while maintaining the integrity of the main chain of the target lipid ion.

[0049] The first dissociation device 3 may include one or more dissociation devices selected from the group consisting of a collision-induced dissociation (CID) device, a surface-induced dissociation (SID) device, an electron transfer dissociation (ETD) device, an electron capture dissociation (ECD) device, an electron collision or collision dissociation device, a photoinduced dissociation (PID) device, a laser-induced dissociation device, an infrared radiation-induced dissociation device, an ultraviolet radiation-induced dissociation device, a nozzle-separator interface dissociation device, an in-source dissociation device, an in-source collision-induced dissociation device, a thermal or temperature source dissociation device, an electric field-induced dissociation device, a magnetic field-induced dissociation device, an ion-ion reactive dissociation device, an ion-molecule reactive dissociation device, an ion-atom reactive dissociation device, an ion metastable ion reactive dissociation device, an ion metastable molecule reactive dissociation device, and an electron ionization dissociation (EID) device.

[0050] In this embodiment, the first dissociation device 3 is a simpler in-source collision-induced dissociation device that applies a voltage through a vacuum interface, for example, to an orifice. The voltage applied to the end electrode is 10-70 eV. This voltage range allows for efficient removal of the polar head groups of phospholipids or sphingolipids.

[0051] <Mass filter> The mass filter 4 executes a mass selection step S4, and selects the target lipid ions whose first chemical bond has been cleaved based on the mass number, to obtain fragment ions.

[0052] The mass filter 4 may comprise one or more mass filters selected from the group consisting of a quadrupole mass filter, a 2D or linear quadrupole ion trap, a Paul or 3D quadrupole ion trap, a Penning ion trap, an ion trap, a magnetic sector mass filter, a time-of-flight mass filter, and a Wien filter.

[0053] <Second dissociation device> The second dissociation device 5 performs a second dissociation step S5 to further dissociate the fragment ions and cleave second chemical bonds of the fragment ions, the bond energy of which is higher than that of the first chemical bond, to obtain diagnostic ions.

[0054] The second dissociation device 5 may include one or more dissociation devices selected from the group consisting of a collision-induced dissociation (CID) device, a surface-induced dissociation (SID) device, an electron transfer dissociation (ETD) device, an electron capture dissociation (ECD) device, an electron collision or collision dissociation device, a photoinduced dissociation (PID) device, a laser-induced dissociation device, an infrared radiation-induced dissociation device, an ultraviolet radiation-induced dissociation device, a nozzle-separator interface dissociation device, an in-source dissociation device, an in-source collision-induced dissociation device, a thermal or temperature source dissociation device, an electric field-induced dissociation device, a magnetic field-induced dissociation device, an ion-ion reactive dissociation device, an ion-molecular reactive dissociation device, an ion-atom reactive dissociation device, an ion metastable ion reactive dissociation device, an ion metastable molecular reactive dissociation device, and an electron ionization dissociation (EID) device.

[0055] Preferably, the second dissociation device 5 is a collision-induced dissociation device with a dissociation energy of 30-70 eV, which can selectively cleave the glycerin skeleton and the aziridine ring, reduce the generation of impurity ions, improve the spectral peak intensity of diagnostic ions, and simplify the spectrogram, making it easier to interpret.

[0056] <Mass spectrometer> The mass spectrometer 6 executes a mass analysis step S6 to perform mass analysis on the diagnostic ions.

[0057] The mass spectrometer 6 may comprise one or more mass spectrometers selected from the group consisting of a 2D or linear quadrupole mass spectrometer, a Paul or 3D quadrupole mass spectrometer, a Penning trap mass spectrometer, an ion trap mass spectrometer, a magnetic sector mass spectrometer, an ion cyclotron resonance (ICR) mass spectrometer, a Fourier transform ion cyclotron resonance (FTIR) mass spectrometer, an electrostatic mass spectrometer arranged to generate an electrostatic field having a quadrupole logarithmic potential distribution, a Fourier transform electrostatic mass spectrometer, a Fourier transform mass spectrometer, a time-of-flight mass spectrometer, an orthogonal acceleration time-of-flight mass spectrometer and a linear acceleration time-of-flight mass spectrometer. Preferably, the mass spectrometer 6 is a high-resolution mass spectrometer such as a time-of-flight mass spectrometer.

[0058] Although the components of the ion mobility spectrometry tandem mass spectrometer of this embodiment have been described above, the present invention is not limited to these. In other embodiments of the present invention, a separation device may be installed upstream of the ion source 1. The separation device may be one or more of liquid chromatography, gas chromatography, supercritical chromatography, capillary electrophoresis, and paper chromatography.

[0059] <Derivatization reaction> The method for identifying the chemical structure of lipids according to this embodiment further includes a derivatization step of labeling carbon-carbon double bonds by a derivatization reaction prior to the ionization step S1. The derivatization step can be performed offline, such as by an experimenter in a laboratory. Alternatively, it can be performed online, such as by automatically introducing a sample and reaction reagents into a reactor to complete the reaction. This embodiment is not limited thereto.

[0060] The derivatization reaction may be any derivatization reaction capable of converting a carbon-carbon double bond into a group that easily dissociates, such as an aziridination reaction, an epoxidation reaction, a singlet oxygen-ene reaction, etc. More specifically, the reaction may be, for example, a Pattern-Burchi reaction, a Diels-Alder reaction, an aza-Prilezhaev reaction, a singlet oxygen-ene reaction, etc., and the present embodiment does not limit the type of reaction employed.

[0061] In this embodiment, the derivatization reaction employs the aza-Prilezhaev reaction, the reaction mechanism of which is shown below. [ka]

[0062] The derivatization reagent is a mass-labeled compound dissolved in an acidic solvent. Specifically, the mass-labeled compound is tert-butyl N-tosyloxycarbamate (N-Boc-O-tosylhydroxylamine, CAS: 105838-14-0), and the acidic reagent is hexafluoroisopropanol. The reaction is heated at 20-100°C for at least 10 minutes to aziridinate the carbon-carbon double bond.

[0063] The dissociation energy for cleaving the glycerol backbone or aziridine ring is higher than that for cleaving the polar head group, but lower than that for cleaving other chemical bonds, such as the carbon-carbon bond of an aliphatic chain. Therefore, in the compound obtained from the derivatization reaction, the first chemical bond (e.g., the polar head group) and the second chemical bond (e.g., the glycerol backbone or aziridine ring) that are primarily to be cleaved can be cleaved without the need for applying a large amount of dissociation energy, whether in the first dissociation step S3 or the second dissociation step S5. This avoids the spectrogram from becoming too complex due to the application of a large amount of dissociation energy, while at the same time further improving the signal intensity of the diagnostic ions.

[0064] Additionally, derivatization of the carbon-carbon double bond with more rigid structures such as aziridine rings or epoxies can further enhance the structural differences between different molecules and improve the resolution of ion mobility spectrograms.

[0065] <Ion reaction process> Hereinafter, the method for identifying the chemical structure of lipids in this embodiment will be described, taking as an example a case where the target lipid ion is PC(18:1 / 16:0).

[0066] Figure 4 shows the specific reaction process of PC (18:1 / 16:0) in this embodiment. First, the raw sample is pretreated, i.e., a derivatization step is performed to convert the carbon-carbon double bonds of unsaturated fatty acids in the raw sample into aziridine rings.

[0067] In the ionization step S1, each component molecule is converted into a positively charged sample ion, followed by mass spectrometry in positive ion mode. Here, PC (18:1 / 16:0) contained in the sample is aziridinylated and then ionized into a target lipid ion. The positive ion obtained by hydrogen addition is the target lipid ion with a mass number of 775.6, and the positive ion obtained by sodium addition is the target lipid ion with a mass number of 797.6.

[0068] Next, in the ion mobility separation step S2, the ion mobility spectrometer can separate the target lipids from the sample ions during a specific time period of one analysis cycle and transport them to a downstream stage. Alternatively, the ion mobility spectrometer can be configured in a filtering mode, i.e., the target lipid ions in the sample ions can be continuously selected and transported to a downstream stage.

[0069] In the first dissociation step S3, the target lipid ions are dissociated to remove the phosphocholine group, which is the polar head group of PC (18:1 / 16:0). By removing the phosphocholine group, fragment ions having the 1,3-dioxolane structure shown in Figure 4 are obtained.

[0070] In the second dissociation step S4, the fragment ions having the 1,3-dioxolane structure may be cleaved by 1,3-dioxolane or by the aziridine ring obtained by the derivatization reaction to form multiple diagnostic ions. Some of the diagnostic ions are used to identify the position of the carbon-carbon double bond in the aliphatic chain, i.e., the diagnostic ions at the C=C position shown in FIG. 4. Some of the diagnostic ions are used to identify the sn position in the carbon-carbon double bond, i.e., the diagnostic ions at the sn position shown in FIG. 4. Some of the diagnostic ions are used to identify the information of the aliphatic chain in which the carbon-carbon double bond exists, i.e., the diagnostic ions of the aliphatic chain shown in FIG. 4.

[0071] Figure 5 shows the mass number m / z = 290 or 360 [M + Na + This is a principle diagram explaining why the sn position of a carbon-carbon double bond can be identified from the diagnostic ion [ ].

[0072] As can be seen from Figure 5, when the carbon-carbon double bond is at a different sn position, the fragment ions and diagnostic ions generated by dissociation are different. For PC(16:0 / 18:1) where the carbon-carbon double bond is at the sn-2 position, the mass number is 290 [M+Na + For PC(18:1 / 16:0) with a carbon-carbon double bond at the sn-1 position, the mass number is 360 [M+Na + ] can be distinguished based on the diagnostic ion.

[0073] As can be seen from the above reaction process, the method for identifying the chemical structure of lipids according to this embodiment can simultaneously identify the position of the carbon-carbon double bond in the fatty chain of a phospholipid or sphingolipid, the structure of the fatty chain, and the sn position with a single sample injection, thereby exhibiting excellent analytical efficiency.

[0074] Although some major steps of the method for identifying the chemical structures of lipids have been described above, the method is not limited thereto and in other embodiments of the present invention, the method may further include other steps.

[0075] For example, before the first dissociation step S3 and the second dissociation step S5 are performed, a first pre-scan step may be performed to perform mass analysis on sample ions that have not undergone the first dissociation step S3 and the second dissociation step S5. The first pre-scan step is used to discover target lipid ions. That is, in this embodiment, the mass number is 775 [M+H + ] or 797[M+Na + ] is found, and the target lipid ions are further subjected to isomer determination.

[0076] Furthermore, for example, after the first dissociation step S3 and before the second dissociation step S5, a second prescan step may be performed to perform mass analysis on the sample ions that have undergone only one dissociation. The second prescan step is used to determine an appropriate dissociation energy. This allows the target lipid ions to be dissociated so that only the polar head groups are cleaved, reducing or avoiding the generation of extraneous fragment ions, improving signal intensity, and making the spectrogram easier to interpret.

[0077] <Experimental Results> FIG. 6 is a comparison diagram of measurement results obtained by a general tandem mass spectrometry (MS2) method in the prior art and measurement results obtained by a method according to an embodiment of the present invention.

[0078] In the two mass spectrograms corresponding to the prior art in Figure 6, the upper one is an MS2 spectrogram, and the lower one is a spectrogram obtained by enlarging the range of the upper frame. In the two mass spectrograms corresponding to the measurement results of this embodiment in Figure 6, the upper one is an MS1 ​​spectrogram (the label "pseudo MS2" refers to the combination of the IMS and the first dissociation device 3 as MS1, and the same applies below), and the lower one is an MS2 spectrogram obtained by further dissociating the ion with mass number 592.

[0079] As can be seen from Figure 6, due to interference from polar head groups, the peak intensity of diagnostic ions in the spectrogram of a typical tandem mass spectrometry MS2 is in the range of about 700-800. In contrast, the peak intensity of diagnostic ions obtained by the IMS-CID-MS / MS method of this embodiment can reach the range of 5000-6000, significantly improving the signal intensity (about 7-8 times) and improving the analytical sensitivity.

[0080] The above are merely preferred embodiments of the present invention, and the present invention is not limited thereto. Any modifications, equivalent substitutions, improvements, etc. that do not deviate from the gist of the present invention are included in the technical scope of the present invention. [Explanation of symbols]

[0081] 1-Ion Source 2-Ion Mobility Spectrometer 3-First dissociation device 4-Mass Filter 5-Second dissociation device 6-Mass spectrometer 7-Ion optical device.

Claims

1. A method for identifying the chemical structure of lipids, comprising: an ionization step of ionizing the sample to obtain sample ions; an ion mobility separation step of separating target lipid class ions from the sample ions based on ion mobility; a first dissociation step of dissociating the target lipid ions with a dissociation energy suitable for cleaving a first chemical bond of the target lipid ions; A mass selection step of selecting the target lipid ions from which the first chemical bond has been cleaved based on their mass numbers to obtain fragment ions; a second dissociation step of dissociating the fragment ions to break at least a second chemical bond of the fragment ions, the second chemical bond having a higher bond energy than the first chemical bond, to obtain diagnostic ions; a mass analysis step of performing mass analysis on the diagnostic ions.

2. The method for identifying the chemical structure of lipids according to claim 1, characterized in that the lipids are unsaturated lipids having a carbon-carbon double bond in the fatty chain, and the method is used to identify the position of the carbon-carbon double bond in the fatty chain and the position of sn in the fatty chain.

3. The method for identifying the chemical structure of lipids according to claim 2, further comprising a derivatization step of labeling the carbon-carbon double bond by a derivatization reaction prior to the ionization step.

4. The method for identifying the chemical structure of lipids according to claim 3, wherein the lipids are phospholipids or sphingolipids, the first chemical bond is a bond to a polar head group of the phospholipid or a polar head group of the sphingolipid, and the second chemical bond is a chemical bond obtained by a derivatization reaction of the carbon-carbon double bond.

5. 4. The method for identifying the chemical structure of lipids according to claim 3, wherein the derivatization reaction is an aziridination reaction, an epoxidation reaction, a Paterno-Buchi reaction, a singlet oxygen-ene reaction, or a Diels-Alder reaction.

6. 2. The method for identifying the chemical structure of lipids according to claim 1, wherein the lipids are fatty acyl, glyceride, glycerophospholipid, sphingolipid, sterol ester, pregnenolone lipid, glycolipid or polyketide.

7. The method for identifying the chemical structure of lipids according to claim 1, further comprising a first pre-scan step of performing mass analysis on the sample ions that have not undergone the first dissociation step and the second dissociation step.

8. 2. The method for identifying the chemical structure of lipids according to claim 1, further comprising a second pre-scan step of performing mass analysis on the sample ions that have undergone dissociation only once.

9. An ion mobility spectrometry tandem mass spectrometer, comprising: an ion source for ionizing the sample to obtain sample ions; an ion mobility spectrometer for separating target lipid ions from the sample ions; a first dissociation device for dissociating the target lipid ions, the dissociation energy of which is suitable for breaking first chemical bonds of the target lipid ions; a mass filter that selects the target lipid ions whose first chemical bond has been cleaved to obtain fragment ions; a second dissociation device for dissociating the fragment ions to break at least a second chemical bond of the fragment ions having a higher bond energy than the first chemical bond to obtain diagnostic ions; a mass spectrometer that performs mass analysis on the diagnostic ions.

10. 10. The ion mobility spectrometry tandem mass spectrometer according to claim 9, wherein the ion mobility spectrometer is a U-type ion mobility spectrometer.

11. 11. The ion mobility spectrometry tandem mass spectrometer of claim 10, wherein the U-shaped ion mobility spectrometer operates in a filtering mode.

12. 10. The ion mobility spectrometry tandem mass spectrometer of claim 9, wherein the mass filter is a quadrupole rod or an ion trap.

13. 10. The ion mobility spectrometry tandem mass spectrometer according to claim 9, wherein the mass spectrometer is a time-of-flight mass spectrometer, a Fourier transform mass spectrometer, a quadrupole mass spectrometer, an ion trap mass spectrometer, or a magnetic mass spectrometer.

14. 10. The ion mobility spectrometry tandem mass spectrometer of claim 9, wherein the ion source is an electrospray ionization source, a nanospray ionization source, a desorption electrospray ionization source, an atmospheric pressure chemical ionization source, an atmospheric pressure photoionization source, or a matrix-assisted laser desorption ionization source.

15. 10. The ion mobility spectrometry tandem mass spectrometer according to claim 9, wherein the first dissociation device and / or the second dissociation device is one or more of a high-energy collisional dissociation device, a collision-induced dissociation device, an oxygen attachment dissociation device, a hydrogen attachment dissociation device, an electron capture dissociation device, a radical-directed dissociation device, an ultraviolet light-induced dissociation device, and a charge remote fragmentation device.

16. The ion mobility spectrometry tandem mass spectrometer of claim 15, wherein the first dissociation device is a collision-induced dissociation device with an end electrode voltage of 10-70 eV, and the second dissociation device is a collision-induced dissociation device with a dissociation energy of 30-70 eV.

17. 10. The ion mobility spectrometry tandem mass spectrometer of claim 9, further comprising a separation device installed in front of the ion source, the separation device being one or more of liquid chromatography, gas chromatography, supercritical chromatography, capillary electrophoresis, and paper chromatography.

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